Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

Abstract

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host-immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.

DFTD cells have low levels of intracellular MHC class I and no surface expression of β2m in vitro and in vivo. (A) Western blot of fibroblast and DFTD whole-cell protein probed with MHCI-mAb, with the MHC class I band shown at 40 kDa for a 20-min exposure to X-ray film. (A Lower) A loading control probed with an antibody to β-actin. By Bradford assay for protein, 35 µg from fibroblast lysate and 50 µg from each DFTD lysate were loaded on the gel. (B) Flow cytometry of fibroblast and DFTD cells, with fluorescence intensity on x axis and number of cells (counts) on y axis. Shaded area, stained with the preimmune serum; solid black line, stained with β2m-Ab; dashed black line, stained with β2m-Ab blocked with native devil β2m protein. (C) IHC on serial sections of primary DFTD biopsies from wild devils stained with an antibody to periaxin (a marker specific for DFTD cells, enabling DFTD cells to be distinguished from host devil cells), devil β2m-Ab, and the preimmune rat serum as a negative control for the devil β2m-Ab. Boxes indicate areas shown at 40× magnification, and arrowheads indicate similar positions in the serial sections, pointing toward DFTD cells as defined by periaxin staining. Positive cells for each marker are stained brown; nuclei are stained blue. (Scale bars: 20× magnification, 50 μm; 40× magnification, 20 μm.)

DFTD cells down-regulate mRNA of genes essential for antigen loading and presentation. (A) RT-PCR amplification of RPL13A (ribosomal protein L13A), MHC class I, β2m, TAP1, TAP2, and tapasin from RNA of a fibroblast cell line and DFTD cell lines in Left, DFTD biopsies and matched host spleen samples in Center, and no-cDNA negative control and markers in Right. Amplicons are between 100 and 300 bp. (B and C) RT-qPCR of β2m (gray) and MHC class I (black) gene expression normalized against RPL13A as a housekeeping gene for tumor lines relative to fibroblast cell line (B), and each DFTD biopsy relative to the matched host spleen sample (C). All samples were tested in triplicate.

MHC class I protein is up-regulated in DFTD cells after treatment with the deacetylation inhibitor, Trichostatin A (TSA). (A) RT-PCR amplification of RPL13A, MHC class I, β2m, TAP1, TAP2, and CIITA from RNA of TSA-treated and untreated cells. Amplicons are between 100 and 300 bp. (B) RT-qPCR of TAP2 (white), β2m (gray), and MHC class I (black) in the tumor lines after treatment with TSA. Fold change is relative to the untreated control cells for each cell line and normalized against RPL13A as a housekeeping gene. A standard curve was constructed from fibroblast expression. DFTD samples were tested in triplicate. (C) Western blot of whole-cell protein from treated and untreated DFTD cells, probed with MHCI-mAb, showing MHC class I at 40 kDa. By Bradford assay for protein, 24 µg from lysate of fibroblasts, 25 µg from lysate of untreated DFTD cells, and 20 µg from lysate of treated DFTD cells were loaded on the gel. (D) Flow cytometry to test for surface expression of β2m on TSA-treated DFTD, with fluorescence intensity on x axis and number of cells (counts) on y axis. Shaded area, stained with serum from a preimmunized rat; dashed line, untreated cells stained with β2m-Ab; solid black line, TSA-treated cells stained with β2m-Ab.